Fibroblast Growth Factor Receptor 3 Inhibited By Short-hairpin RN As In Multiple Myeloma And Study Of The Function Of The Receptor

Multiple myeloma (MM) is an aggressive neoplastic disease characterized by the increased proliferation and extended life span of monoclonal plasma cells. The t(4;14)(pl6.3;q32.3) translocation occurs in approximately 10%-20% of MM resulting in ectopic expression of functional FGFR3 . FGFR3 is a membrane spanning tyrosine kinase receptor that has a high affinity for fibroblast growth factors (FGFs) . FGFR3 contains three glycosylated extracellular immunoglobulin-like (Ig-like) domains, a transmembrane domain, and a split intracellular tyrosine kinase domain. Under ligand stimulation, FGFR3 undergoes dimerization and tyrosine autophosphorylation, resulting in cell proliferation or differentiation. Depending on the cellular context, this effect is achieved through different signal transduction pathways. FGFR3 is not expressed or at very low level of expression in B-cell lineage but is overexpressed as a consequence of the translocation. Interestingly, FGFR3 gene mutations associatedwith human skeletal dysplasia have also been identified in some MM with t(4;14). Activating mutations lead to a transforming event suggesting that overexpression of activating mutations of FGFR3 may play an oncogenic role in MM. Thus, FGFR3 could become a specific therapeutic target in patients suffering from myeloma characterized by the presence of 4; 14 translocation.In this study, three shRNAs (small-hairpin RNA) targeting different sites of FGFR3 were selected and subsequently transfected into KMS-11, OPM-2, and NCI-H929 human myeloma cell lines all of which are characterized by t(4;14) and FGFR3 over expression. The combination of these triple shRNAs can effectively inhibit FGFR3 expression in all three cell lines. Sequential immunocytochemistry/FISH was employed to validate that the shRNAs specifically inhibited FGFR3 expression on OPM-2 cells. Decreased expression of BCL2 and MCL1 protein, and increased staining of annexin V positive cells demonstrated that inhibiton of FGFR3 induce apoptosis. After confirming downregulation of FGFR3 by reverse transcriptase (RT)-PCR and quantitative real-time PCR, HU-133 plus 2.0 arrays were employed to compare the gene expression profile of shRNA treated sample with that of the control. Besides downregulation of FGFR3, expression of the antiapoptotic genes CFLAR, BCL2, MCL1 and some members of NFkB family decreased, while expression of the proapoptotic genes CYC, BID, CASP2 and CASP6 increased. Microarray results also revealed changes including genes previously implicated in MM pathogenesis (RAS, RAF, IL6R, VEGF), as well as others (TLR4, KLF4, GADD45 A) not previously linked to MM.Microarray results showed that inhibition of FGFR3 will decrease vascular endothelial growth factor (VEGF) expression in OPM-2 MM cells. This phenomenon also existed in the KMS-11 MM cell line in our previous study. Myeloma cells express and secrete VEGF and fibroblast growth factor, both of which are considered potent angiogenic cytokines and are likely to contribute to the increased angiogenic potential of bone marrow plasma cells in progressive MM.In order to better understand the molecular mechanism of FGFR3 and VEGF, L6 cells lacking any endogenous FGFR were stably transfected with FGFR3 at the present study. The FGFR3 expression was validated by Northern blot and Western blot. Compared with parental L6 cell and L6V (transfected with vector only), L6 cells with stably transfected FGFR3 showed overexpression of VEGF. [3~H] thymidine uptake and CellTiter 96 Aqueous One Solution cell proliferation assay demonstrated that DNA synthesis and cell proliferation were increased in FGFR3 expression L6 cells. These observations indicate that FGFR3 might be associated with regulation of VEGF.Our observations indicate that shRNAs can specifically and effectively inhibited FGFR3 expression, and inhibition of FGFR3 can induce apoptosis and down-regulate VEGF expression. This targeted approach may be worth testing in MM patients with t(4;14) and FGFR3 overexpression in the future.